CA2118207C - Bacillus thuringiensis m200 for the biological control of agricultural and horticultural entomological pests - Google Patents

Abstract

The invention relates to a strain of Bacillus thuringiensis M200, a sample of which has been deposited under the accession number NCIMB 40385, or a recombinant or mutant thereof having entomocidal activity against lepidopterous pests. The invention also relates to the use of the strain, recombinant or mutant in control of insect pests.

Description

CA 02ll8207 l998-04-06 BIOLOGICAL CONTROL OF PESTS
This invention relates to biological methods of pest control and more particularly to methods and compositions utilising Bacillus thuringiensis for the control of agricultural and horticultural pests.
Bacillus thuringiensis is a spore forming bacterium which has long been known to kill insects and many hundreds of isolates (strains) of this species have been listed. The properties of individual B. thuringiensis strains and their activity against insect pests vary widely from strain to strain.
We have now isolated a new strain of Bacillus thuringiensis which has been found to be highly active against agricultural and horticultural pests including those of the family Pieridae, including Pieris brassicae (Large White Butterfly), and pests of the family Yponomeutidae, including Plutella xylostella (Diamond-back moth). The activity of the new strain, designated M200, is several-fold greater towards the above pests than many known strains of B. thuringiensis including those used commercially and those under current development which have been described in the literature.
The present invention comprises Bacillus thuringiensis strain M200 a sample of which has been deposited under the Budapest Treaty with the National Collection of Industrial and Marine Bacteria under Accession No. NCIMB
40385, on 20 March 1991.
The present invention also comprises the use of B.
thuringiensis strain M200 for the control of pests which affect plants. Accordingly the invention comprises a method of pest control in which agricultural and horticultural produce are protected against pests by application of this new strain to the growing plants or other horticultural products or to their growing locations. The invention also comprises compositions containing the new strain and formulated for agricultural or la horticultural app' ~rticln. The invention is applicable also to variants, mutants and recombinants of the new strain which are similarly biologically effective.

A recombinant of strain M200 is one which is produced when the strain has been used as a recipient for additional genetic material in the form of an additional plasmid. For example, the additional plasmid can be transferred by plasmid transfer (conjugation) from another Bt strain, or the additional plasmid can contain eg a cloned toxin gene prepared by recombinant DNA
techniques and inserted into the recipient strain by transformation using techniques well known to those skilled in the art. In both cases, the recipient strain retains its original genetic material and thus the same insecticidal activity of the original strain is maintained. Of course, additional or enhanced insecticidal activity may be present in the recombinant strain by virtue of the added genetic material.

The invention will now be further described.

The strain Bacillus thuringiensis M200 has the H-serotype 3a3b. When grown for 24h at 30~C
on nutrient agar the colonies are circular, entire, convex, off-white, have a ground glass appearance and a diameter of 2-8mm. The vegetative cells are Gram-positive aerobic rods producing sub-terminal spores towards the end of the growth cycle. At the same time as sporulation, a parasporal, proteinaceous crystal is produced which is approximately the same size as the spore.

2 ~ 7 biochemical profile of M200 on standard substrates (API ZONE test strip) is shown below.
HD1 is a commercially available strain.

Strain Code No.
Reaction NO3 (nitrate reduction) +
TRP (indole production) GLU (glucose acidification) ADH (arginine dihydrolase) +
URE (urease) ESC (esculin hydrolysis) +
GEL (gelatine hydrolysis) + +
PNPG (,~-glactosidase) GLU (glucose assimilation) + +
ARA (arabinose assimilation) MNE (mannose assimilation) MAN (mannitol assimilation) NAG (N-acetyl glucosamine assimilation) + +
MAL(maltose assimilation) + +
GNT (gluconate assimilation) + +
CAP (caprate assimilation) ADI (adipate assimilation) MLT (malate assimilation) + +
CIT (citrate assimilation) PAC (phenylacetate assimilation) * Trade - ma rk B

2118~07 B. thuringiensis strains produce a number of proteins which are toxic to insects. There is an internationally recognised classification for these toxins. By the use of specific DNA probes it is possible to determine which toxin genes a particular strain possesses. The major toxin gene content of strains HD1 and M200 is compared below.

HD1: CRY 1A(a), CRY 1A(b), CRY 1A(c), CRYIIA
M200: CRY 1A(a), CRY 1 A(b) [not expressed], CRY 1A(c), CRY 1 E(c), CRYIIA

B. thuringiensis can be produced on a large scale by submerged fermentation using processes such as those described by Lee et al in Microbial Biomass [1979, A.H. Rose (ed). Academic Press pp 91-114]. Typical growth media may contain any of the following ingredients in various combinations: fishmeal, cottonseed meal, soybean meal, molasses, diammonium phosphate, starch. During production by fermentation, after normal growth of Bacillus thuringiensis, the mother cells Iyse and release the spores and crystals into the growth medium. The spores and crystals may be harvested by centrifugation or filtration, spray drying, vacuum drying, or a method of precipitation, such as the lactose coprecipitation technique as reported by Dulmage etal(Joumal of Invertebrate Pathology, 15, 15-20, 1970). The resulting spore-crystal complex is stable for long periods and can be formulated into a product suitable for application to crops.

The invention also includes the use of delta endotoxins derived from the above-mentioned new organism. The invention provides an entomocidal substance derived from Bacillus thuringiensis strain M200, or from a recombinant or mutant thereof. In one embodiment the entomocidal substance is a spore-crystal complex. The spore-crystal complex or the composition containing it may be administered to the plants or crops to be protected together with certain other insecticides or chemicals without loss or potency.

It is possible to kill the spores in the spore-crystal (eg by gamma irradiation), or to avoid producing spores by use of an asporogenous crystaliferous mutant, thereby producing a non-viable product. A non-viable product may be advantageous in certain circumstances where it is desired to prevent the spread of bacteria for aesthetic reasons or to avoid causing disease in beneficial insects. However, non-viable products are generally not as active as those containing live spores and as a further disadvantage there is the increased cost of killing the spores.

The invention further provides an entomocidal substance derived from Bacillus thuringiensis strain M200 (NCIMB 40385) or a recombinant or mutant thereof, or an entomocidal substance as defined above together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant. The composition may also contain a further biologically active compound selected from fertilisers, micronutrient donors, plant growth preparations, herbicides, insecticides, fungicides, bactericides, nematicides and molluscicides and mixtures thereof. The composition may comprise from 0.1 to 99% by weight of Bacillus thuringiensis M200 or the recombinant or mutant thereof, or the entomocidal substance; from 1 to 99.9% by weight of a solid or liquid adjuvant and from 0 to 25% by weight of a surfactant.

The invention in addition provides a method of combatting pests which comprises applying to the pests or to their environment an entomocidally effective amount of Bacillus thuringiensis strain M200, or a I~COl 1 Ib-. ,ant or mutant thereof, or an entomocidal substance as defined above, or a composition containing said strain, recombinant, mutant or substance.

Bacillus thuringiensis strain M200 or the compositions containing it may be administered to the plants or crops to be protected together with certain other insecticides or chemicals without loss of potency. It is compatible with most other commonly used agricultural spray materials but should not be used in extremely alkaline spray solutions. It may be administered as a dust, a ~118207 suspension, a wettable powder or in any other material form suitable for agricultural application.

The invention furthermore relates to a method of treating crops which comprises applying an entomocidally effective amount of B. thuringiensis M200, or a composition thereof.

Bacillus thuringiensis M200, is normally applied in the form of compositions and can be applied to the crop area to be treated, simultaneously or in succession, with further biologically active compounds. These compounds may be both fertilisers or micronutrient donors or other preparations that influence plant growth. They may also be selective herbicides, insecticides, fungicides, bactericides, nemAticides, ~ uscicWes or mixtures of several of these preparations, if desired, together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, eg natural or regenerated mineral substances, solvents, dispersants, wetting agents, takifiers, binders or fertilisers.

The fommulations, ie the compositions, preparations or mixtures containing B. thuringiensis M200 as an active ingredient or combinations thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner eg by homogeneously mixing and/or grinding the active ingredients with extenders eg solvents, solid carriers and in some cases surface-acting compounds (surfactants).

Suitable solvents are: aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, eg xylene mixtures or substituted napthalenes, phthalates such as dibutylphthalate or dioctylphthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols, such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, ~118207 dimethylsufoxide or dimethylformamide as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil; or water.

The solid carriers used eg for dusts and dispersable powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, eg especially dolomite or pulverised plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants.

Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-acting compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), eg the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained, eg from coconut oil or tallow oil. Further suitable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids. More frequently, however, so-called surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylaryl-sulfonates.

The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted a,~ ,on -lrn salts and generally contain a C8-C22 alkyl radical which also includes the alkyl moiety of acyl radicals, eg the sodium or calcium salt of lignosulfonic acid, or dodecylsufate, or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms.
Examples of alkyl-arylsufonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapththalenesulfonic acid or of a naphthalenesulfonic acid/fo",~ldehyde condensation product. Also suitable are corresponding phosphates, eg salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylene sorbitan-trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as N-substituent, at least one C8-Cz alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates eg stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described eg in "McCutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp, Ridgewood, New Jersey, 1979; Dr Helmut Stache, "Tensid Taschenbuch" (Handbook of Surfactants), Carl Hanser Verlag, MunichNienna.

The entomocidal compositions usually contain 0.1 to 99%, preferably 0.1 to 95% of Bacillus thuringiensis M200, or combination thereof with other active ingredients, 1 to 99.9% of a solid or liquid adjuvant and 0 to 25%, preferably 0.1 to 20%, of a surfactant.

Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations of substantially lower concentration.

The compositions may also contain further ingredients, such as stabilisers, antifoams, viscosity regulators, binders, tackifiers as well as fertilisers or other active ingredients in order to obtain special effects.

The invention will now be further described with reference to experimental and field test data obtained in comparative testing of isolate M200 and of other B. thuringiensis isolates.

~118207 FY~nple 1 - Large Scale Culture of B. thuringiensis Bacillus thuringiensis strain M200 can be cultured on a large scale as follows:

The strain is maintained in Iyophilized ampoules, which are used as stocks for inoculating the strain onto agar slopes. The slopes are incubated for 1 to 5 days at 20 to 40~C, preferably 25 to 33~C, following which they are used to inoculate shaken flasks containing an aqueous culture medium. Typical growth media might contain the following ingredients in various combinations:

3-5% fishmeal, 2-3% cottonseed meal, 2-3% soybean meal, 2-4% molasses, 1% diammonium phosphate and 0.5-1% starch. The flasks are shaken at a temperature of 20 to 40~C preferably 30~C for 1 to 5, preferably 1 to 2 days (this vegetative growth stage can be repeated optionally at least once in a separate flask), and used to inoculate a preculture fermenter containing an aqueous cultivating medium. The fermentation medium containing the inoculate is stirred and aerated at a temperature of 20 to 40~C, preferably 30 to 35~C, (optionally this preculture fermentation stage can be repeated at least once in a separate larger container), before being introduced at 2 to 20 per cent by weight of the incubating liquor into a production fermenter, containing an aqueous cultivating medium. The medium is stirred and aerated at a temperature of 20 to 40~C, preferably 30 to 35~C until the Bacillus thuringiensis M200 broth is harvested when sporulation and crystal production in the production fermenter reaches a maximum. The agar and broth used for preparation of inocula for the preculture fermenter should contain at least one nitrogen source, at least one carbon source and at least one salt, preferably peptone, glucose and at least one salt. The fermentation media should contain at least one nitrogen source (eg peptone, yeast extract, corn steep liquor, soya bean meal, cotton seed meal, fishmeal), at least one carbohydrate source (eg glucose, lactose, sucrose, starch or raw material rich in these constituents) at at least one mineral salt. The nitrogen and carbohydrate should be balanced to exhaust as near as possible simultaneously. Cultures can be concentrated after harvesting by centrifugation to produce a slurry containing a spore/crystal concentrate. The concentrate can be converted into a dry powder by spray-drying.

Example 2 - Insect Bioassays The activity of B. thuringiensis strain M200 against the insects Pieris brassicae and Plutella xylostella was compared in bioassays with the commercially available strain HD1. Bacterial powders were prepared as described in Example 1 and activities against insects assayed by the addition of a series of concentrations of the powders to artificial diets on which the larvae fed.
For P. brassicae the semi-synthetic diet of David, W.A.L. and Gardiner, R.O.C. (1965), [Nature, London 207, No 4999, pp 882-883] was used. All larvae used in bioassays were neonates.
Mortality was recorded after 5 days with the temperature maintained at 25~C. For P. xylostella the same semi-synthetic diet was used, except that all antibiotics were omitted, but all larvae used in bioassays were at the beginning of the third instar. Mortality was recorded after 5 days with the temperature maintained at 25~C.

It can be seen from the results expressed in Table 1 that the activity of B. thuringiensis strain M200 towards P. brassicae is 2.5 to 4-fold greater than that of HD1. Similarly the activity of B.
thuringiensis strain M200 towards P. xylostella is 1 6-fold greater than that of HD1.

- ~118207 Example 3 - Formulation exa"--ples for solid active ingredients of Bacillus thurin~iensis M200 or cG~IlJinations thereof with other active ingredients (throughout, percentages are by weight) 1. Wettable powders a) b) c) Bacillus thuringiensisM200 25% 50% 75%
sodiumlignosulfonate 5% 5%
sodium laurylsulfate 3% - 5%
sodium diisobutylnapthalenesulfonate - 6% 10%
octylphenol polyethylene glycol ether (7-8 moles of ethylene oxide) - 2%
highly dispersed silicic acid 5% 10% 10%
kaolin 62% 27%

Bacillus thunngiensis M200, is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentration.

2. Fmulsifiable concentrate Bacillus thuringiensis M200 10%
octylphenol polyethylene glycol ether (4-5 moles of ethylene oxide) 3%
calcium dodecylbenzenesulfonate 3%
castor oil polyglycol ether (36 moles of ethylene oxide) 4%
cyclohexanone 30%
xylene mixture 50%

Emulsions of any required concentration can be obtained from this concentrate by dilution with water.

3. Dusts a) b) Bacillus thuringiensisM200 5% 8%
talcum 95%
kaolin - 92%

Ready for use dusts are obtained by mixing the active ingredient with the carriers and grinding the mixture in a suitable mill.

2I18~7 4. Extruder Qranulate Bacillus thuringiensis M200 10%
sodium lignosulfonate 2%
carboxymethylcellulose 1 %
kaolin 87%

The active ingredient or combination is mixed and ground with the adjuvants and the mixture is subsequently moistened with water. The mixture is extruded, granulated and then dried in a stream of air.

5. Coated granule Bacillus thuringiensis M200 3%
polyethylene glycol (mol. wt. 200) 3%
kaolin 94%

The finely ground active ingredient or combination is uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner.

2t.I8207 6. Suspension Concentrate Bacillus thuringiensis M200 40%
ethylene glycol 10%
nonylphenol polyethylene glycol ether (15 moles of ethylene oxide) 6%
sodium lignosulfonate 10%
carboxymethylcellulose 1 %
37% aqueous formaldehyde solution 0.2%
silicone oil in the form of a 75% aqueous emulsion 0.8%
water 32%

The finely ground active ingredient or combination is intimately mixed with the adjuvants giving a suspension concer,l,ale from which suspensions of any desired concentration can be obtained by dilution with water.

Example 4 - Field Trials Bacillus thuringiensis strain M200 was prepared as a wettable powder formulation using the method described in Example 3. This fommulation contained 25% by weight of strain M200. The strain was tested for control of P. xylostella, Pieris rapae and Trichoplusia ni in a series of field trials on cabbages. The results, expressed as percentage control of insect larvae, are summarised in Table 2. These results clearly indicate that, in the field, strain M200 is highly active against the insect pests tested. The wettable powder used in these trials represents only one example of an effective formulation, preparation of many other effective formulations will be known to those skilled in the art.

~1 18207 Table 1 Activity of Bacillus thurinyiensis M200 against Pieris brassicae and Plutella xylostella LC50 of Isolate Isolate (Fiducial limits) P. bra-ssie~e M200 0.16 (0.07 - 0.26) M200 (replicate) 0.096 (0.038 - 0.17) HD1 0.40 (0.30 - 0.50) P. xylostella M200 0.07 (0.014 - 0.50) HD1 1 .32 (0.95 -1 .83) LC50 is expressed as the dose of B. thuringiensis powder (~g per g diet) which gave 50%
mortality.

Table 2 Activity of B. thuri"giensis strain M200 in field trials against Plutella xylostella, Pieris rapae and Trichoplusia nion caL,~age Application Rate % Control Strain (Ib/acre) P. xylostella T. ni P. rapae 0.25 84.3 - 100.0 0.50 93.9 - 97.0 M200' 1.00 95.0 83.5 100.0 1.50 - 93 7 Wettable powder containing 25% B. thuringiensis strain M200 and prepared as described in Example 3.

Claims (8)

1. A strain of Bacillus thuringiensis M200, a sample of which has been deposited under the accession number NCIMB
40385, or a recombinant or mutant thereof having entomocidal activity against lepidopterous pests.

2. An entomocidal substance derived from Bacillus thuringiensis strain M200 (NCIMB 40385), or from a recombinant or mutant thereof, the entomocidal substance being a spore-crystal complex.

3. The use of Bacillus thuringiensis strain M200 (NCIMB
40385), or a recombinant or mutant thereof, for control of insect pests susceptible to M200 including insect pests selected from the group consisting of Plutella xylostella, Pieris rapae, Pieris brassicae and Trichoplusia ni.

4. An entomocidal composition comprising Bacillus thuringiensis strain M200 (NCIMB 40385), or a recombinant or mutant thereof, or an entomocidal substance according to claim 2, together with a carrier, diluent, surfactant or application-promoting adjuvant.

5. An entomocidal composition according to claim 4 in the form of a wettable powder comprising from 25 to 75% by weight of Bacillus thuringiensis M200, or a recombinant or mutant thereof, or an entomocial substance derivable from Bacillus thuringiensis strain M200 (NCIMB 40385), or from a recombinant or mutant thereof, said entomocidal substance being a spore-crystal complex, from 0 to 5% by weight of sodium lignosulfate, from 0 to 5% by weight of sodium laurylsulfate, from 0 to 10% by weight of sodium diisobutylnapthalene sulfonate, from 0 to 2% by weight of octylphenol polyethylene glycol ether (7-8 moles of ethylene oxide), from 5 to 10% by weight of highly dispersed silicic acid; and from 0 to 62% by weight of kaolin.

- 18a -

6. A composition according to claim 4, also containing a further biologically active compound selected from fertilisers, micronutrient donors, plant growth preparations, herbicides, insecticides, fungicides, bactericides, nematicides and molluscicides and mixtures thereof.

7. A composition according to claim 4, comprising from 0.1 to 99% by weight of Bacillus thuringiensis M200 or a recombinant or mutant thereof, or an entomocidal substance according to claim 2, from 1 to 99% by weight of a solid or liquid adjuvant; and from 0 to 25% by weight of a surfactant.

8. A method of combatting lepidopterous pests which comprises applying to the pests or to their environment an entomocidally effective amount of Bacillus thuringiensis strain M200 (NCIMB 40385), or a recombinant or mutant thereof, or an entomocidal substance according to claim 2, or a composition containing said strain, recombinant, mutant or substance.